Method for nucleotide detection

ABSTRACT

A method of inhibiting light-induced degradation of nucleic acids includes irradiating a portion of the nucleic acids in the presence of a detection solution comprising a polyphenolic compound. A method of detecting a nucleic acid having a fluorescent tag includes irradiating at least a portion of the nucleic acid with light of a suitable wavelength to induce a fluorescence emission and detecting the fluorescence emission. Optionally, the polyphenolic compound is gallic acid, a lower alkyl ester thereof, or mixtures thereof. A kit includes one or more nucleotides, an enzyme capable of catalyzing incorporation of the nucleotides into a nucleic acid strand and a polyphenolic compound suitable for preparing a detection solution.

The present invention relates generally to nucleic acid detection, andmore specifically to iterative nucleic acid detection.

BACKGROUND OF THE INVENTION

Numerous methods used to detect and characterize nucleic acid structuresemploy tagging schemes that rely on electromagnetic radiation (EM)emission of an excited state light-absorbing chromophore. Examples ofsuch photoluminescent processes include phosphorescence and fluorescenceemission. Fluorescence detection, for example, has been used in DNAsequencing to great effect due, in part, to the high degree ofsensitivity allowing single molecule detection.

Performing iterative fluorescent detection steps in an array context,such as sequencing by synthesis, can cause fluorescence signal intensityloss (see, for example, Fedurco et al. WO2006/064199). This problem wasaddressed, in part, by the addition of ascorbate to a detection solutionto increase the number of useful detection cycles from about eight toten cycles, in the absence of ascorbate, to about 25 cycles in thepresence of ascorbate. The possible mechanisms that underlie this signalloss are numerous, and can include cleavage of individual nucleic acidmembers from the support.

There are a number of pathways by which nucleic acid damage can occurduring irradiation in fluorescence detection. Fluorescence emissionnormally occurs with the emission of light of a longer wavelength (lowerenergy), than the original irradiating source. However, under conditionsin which intense EM radiation is being absorbed by the fluorophore, suchas in laser-induced fluorescence (LIF), it is possible for a molecule toabsorb two photons, which can lead to the emission of higher energyradiation of smaller wavelengths than the original excitation source.This multiple photon absorption can cause the fluorophore to emit EMradiation in the UV-visible region which can contribute to nucleic acidbase dimerization and/or the generation of reactive oxygen species.

For example, it has been indicated that exposure of whole cells toultraviolet (UV) radiation can cause DNA damage via the directphotochemical [2+2] photocycloaddition reaction of thymine or cytosineto provide cyclobutane pyrimidine dimers, such as TT, TC, and CC. Suchdirect photocycloaddition reactions can occur in the UV B and UV Cregions which extend from about 100 nm to about 315 nm.

In the UV A region through a portion of the visible region, spanningfrom about 315 nm to about 500 nm, a complex mixture of indirectmechanisms can also cause DNA damage through photosensitization of othercellular components. Such indirect mechanisms can result in pyrimidinedimer formation and oxidative DNA modification via reactive species suchas singlet oxygen, superoxide anion, and iron-promoted hydroxyl radicalformation. Finally, it also has also been indicated that reactivesinglet oxygen can be generated by fluorescence quenching of an excitedstate fluorophore by triplet oxygen. Any combination of direct orindirect pyrimidine dimerization and nucleic acid damage due to variousreactive oxygen species observed in whole cells can be the underlyingcause of fluorescence signal intensity loss observed in the arraycontext.

There is a need to further reduce fluorescent signal intensity loss forapplications in sequencing by synthesis to facilitate sequencing of longnucleotide sequences, including sequences of 50, 75, 100, 200, and 500nucleotides or more. Moreover, solutions to fluorescent signal intensityloss in the context sequencing by synthesis are readily applicable toother nucleic acid detection platforms that employ multiple irradiationsteps. The present invention satisfies this need and provides relatedadvantages as well.

SUMMARY OF THE INVENTION

Provided herein is a method of inhibiting light-induced degradation ofnucleic acids. The method includes irradiating a portion of said nucleicacids in the presence of a detection solution comprising a polyphenoliccompound. Also provided is a method of detecting a nucleic acid having afluorescent tag. The method includes irradiating at least a portion ofsaid nucleic acid with light, wherein said light comprises a suitablewavelength to induce a fluorescence emission, detecting the fluorescenceemission. Optionally, the polyphenolic compound is gallic acid, a loweralkyl ester thereof, or mixtures thereof.

In addition, provided herein is a kit comprising one or morenucleotides, an enzyme capable of catalyzing incorporation of thenucleotides into a nucleic acid strand and a polyphenolic compoundsuitable for preparing a detection solution.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a graphic representation of the relative effectiveness ofrepresentative polyphenolic compounds, and related structures, inproviding protection from light-induced degradation. Increasedeffectiveness is shown from top (least) to bottom (most).

FIG. 2 shows a graphic representation of the relative effectiveness ofrepresentative polyphenolic compounds, and related structures, inproviding protection from light-induced degradation. Increasedeffectiveness is shown from top (least) to bottom (most).

FIG. 3 shows a graph plotting error versus cluster passing filter (PF)number.

FIG. 4 shows coverage plots in a control sequence, in the presence ofgallic acid, in the presence of urea, and in the presence of gallic acidand urea, over 75 cycles.

FIG. 5 shows coverage plots in a control sequence, in the presence ofgallic acid, in the presence of urea, and in the presence of gallic acidand urea, over 100 cycles.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed, in part, to a method of inhibitinglight-induced degradation of nucleic acids during a detection step thatincludes irradiating a portion of the nucleic acids in the presence of adetection solution having a polyphenolic compound. The presence of thepolyphenolic compound in the detection solution inhibits the amount oflight-induced degradation of the nucleic acids.

The present invention is also directed, in part, to a method ofdetecting a nucleic acid having a fluorescent tag that includes a)irradiating the nucleic acid with light having a suitable wavelength toinduce a fluorescence emission; b) detecting the fluorescence emission;and repeating these steps iteratively. The irradiating step is carriedout in the presence of a detection solution that includes a polyphenoliccompound and serves to reduce light-induced degradation of the nucleicacid.

Polyphenolic compounds, exemplified in FIGS. 1 and 2 and Tables 1 and 2,used in detection solutions provide protection to nucleic acids fromlight-induced degradation that can occur during detection stepsperformed in various assays, including for example, base calling insequencing by synthesis. The polyphenolic compounds disclosed hereinhave been identified as successfully ameliorating the effects oflight-induced degradation among numerous other classes of compounds thatcan shut down plausible mechanistic degradation pathways. Such compoundsclasses include, without limitation, hydroxyl radical quenchers,reactive oxygen species (singlet oxygen, superoxide anion) quenchers,oxygen scavengers, triplet state quenchers, and hole quenchers.

In some embodiments, the presence of urea, with or without ascorbate, inthe detection solution was found to further reduce the amount oflight-induced degradation of the nucleic acids when used with apolyphenolic compound. Table 2 and FIGS. 4 and 5, show this synergy inthe presence of urea and ascorbate. While urea has a significant impacton reducing light-induced degradation when used in conjunction with apolyphenolic compound, using urea alone has only a minor impact withrespect to any such protective benefits.

Methods of the invention that employ a detection solution having apolyphenolic compound protect the integrity of nucleic acids when theyare exposed to repeated and/or intense irradiation, as might be employedin a variety of contexts, including without limitation, high throughputor rapid sequencing techniques such as, sequencing by synthesis andsequencing by ligation, nucleic acid microarray detection techniquessuch as gene chips and DNA microarrays, and quantitative polymerasechain reaction (Q-PCR) techniques such as real time polymerase chainreaction (PCR) and multiplex PCR. As described above, the presence ofurea can enhance the protective role of the polyphenolic compound in anyof these contexts. In the Examples disclosed herein, the use of thedetection solution in sequencing by synthesis in an array format, inparticular, lowered fluorescent signal decay and provided lower errorrates over 50 to 120 cycles of repeated detection steps.

The detection solutions, which can be provided in kit form, can also beused in fluorescence detection techniques that have been employed inmechanistic biochemistry. For example, fluorescence polarization hasbeen indicated as a powerful technique in studying molecularinteractions, including without limitation, receptor-ligandinteractions, such as hormone-receptor interactions, protein-peptideinteractions, and DNA-protein interactions. For example, Singleton etal. Tetrahedron 63(17): (2007) incorporated a fluorescent guanine analoginto oligonucleotides in studying RecA protein interactions with DNA.The detection solution employed in the methods disclosed herein can beused in real-time kinetic measurements where fluorophores are employed.The protective effects against light-induced degradation afforded by thedetection solution need not be limited to the protection of nucleicacids. Thus, for example, the detection solution can also be used toprotect the integrity of proteins, peptides, carbohydrates, and smallmolecules, any of which can be susceptible to reactive oxygen species,or the like, generated under conditions for measuring fluorescenceemission.

As used herein, the term “nucleic acid” is intended to mean at least twonucleotides covalently linked together. Nucleic acid encompasses theterm oligonucleotide, polynucleotide, and their grammatical equivalents.A nucleic acid of the present invention will generally containphosphodiester bonds, although in some cases nucleic acid analogs canhave alternate backbones, comprising, for example, phosphoramide(Beaucage et al., Tetrahedron 49(10): 1925 (1993) and referencestherein; Letsinger, J. Org. Chem. 35:3800 (1970); Sprinzl et al., Eur.J. Biochem. 81:579 (1977); Letsinger et al., Nucl. Acids Res. 14:3487(1986); Sawai et al, Chem. Lett. 805 (1984), Letsinger et al., J. Am.Chem. Soc. 110:4470 (1988); and Pauwels et al., Chemica Scripta 26:14191986)), phosphorothioate (Mag et al., Nucleic Acids Res. 19:1437(1991); and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu et al.,J. Am. Chem. Soc. 111:2321 (1989), O-methylphophoroamidite linkages (seeEckstein, Oligonucleotides and Analogues: A Practical Approach, OxfordUniversity Press), and peptide nucleic acid backbones and linkages (seeEgholm, J. Am. Chem. Soc. 114:1895 (1992); Meier et al., Chem. Int. Ed.Other analog nucleic acids include those with positive backbones (Denpcyet al., Proc. Natl. Acad. Sci. USA 92:6097 (1995); non-ionic backbones(U.S. Pat. Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and4,469,863; Kiedrowshi et al., Angew. Chem. Intl. Ed. English 30:423(1991); Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); Letsingeret al., Nucleoside & Nucleotide 13:1597 (1994); Chapters 2 and 3, ASCSymposium Series 580, “Carbohydrate Modifications in AntisenseResearch”, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker et al.,Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffs et al., J.Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743 (1996)) andnon-ribose backbones, including those described in U.S. Pat. Nos.5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580,“Carbohydrate Modifications in Antisense Research”, Ed. Y. S. Sanghuiand P. Dan Cook. Nucleic acids containing one or more carbocyclic sugarsare also included within the definition of nucleic acids (see Jenkins etal., Chem. Soc. Rev. (1995) pp 169-176). Several nucleic acid analogsare described in Rawls, C & E News Jun. 2, 1997 page 35. All of thesereferences are hereby expressly incorporated by reference. Thesemodifications of the ribose-phosphate backbone may be done to facilitatethe addition of labels, or to increase the stability and half-life ofsuch molecules in physiological environments.

A nucleic acid of the present invention will generally contain aspecific sequence of four nucleotide bases: adenine (A); cytosine (C);guanine (G); and thymine (T). Uracil (U) can also be present, forexample, as a natural replacement for thymine when the nucleic acid isRNA. Uracil can also be used in DNA. A nucleic acid used in theinvention can also include native or non-native bases. In this regard, anative deoxyribonucleic acid can have one or more bases selected fromthe group consisting of adenine, thymine, cytosine or guanine and aribonucleic acid can have one or more bases selected from the groupconsisting of uracil, adenine, cytosine or guanine. It will beunderstood that a deoxyribonucleic acid used in the methods orcompositions set forth herein can include uracil bases and a ribonucleicacid can include a thymine base. Exemplary non-native bases that can beincluded in a nucleic acid, whether having a native backbone or analogstructure, include, without limitation, inosine, xathanine,hypoxathanine, isocytosine, isoguanine, 2-aminopurine, 5-methylcytosine,5-hydroxymethyl cytosine, 2-aminoadenine, 6-methyl adenine, 6-methylguanine, 2-propyl guanine, 2-propyl adenine, 2-thioLiracil,2-thiothymine, 2-thiocytosine, 15-halouracil, 15-halocytosine,5-propynyl uracil, 5-propynyl cytosine, 6-azo uracil, 6-azo cytosine,6-azo thymine, 5-uracil, 4-thiouracil, 8-halo adenine or guanine,8-amino adenine or guanine, 8-thiol adenine or guanine, 8-thioalkyladenine or guanine, 8-hydroxyl adenine or guanine, 5-halo substituteduracil or cytosine, 7-methylguanine, 7-methyladenine, 8-azaguanine,8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine,3-deazaadenine or the like. A particular embodiment can utilizeisocytosine and isoguanine in a nucleic acid in order to reducenon-specific hybridization, as generally described in U.S. Pat. No.5,681,702.

A non-native base used in a nucleic acid of the invention can haveuniversal base pairing activity, wherein it is capable of base pairingwith any other naturally occurring base. Exemplary bases havinguniversal base pairing activity include 3-nitropyrrole and5-nitroindole. Other bases that can be used include those that have basepairing activity with a subset of the naturally occurring bases such asinosine, which basepairs with cytosine, adenine or uracil.

As used herein the term “array of nucleic acids” means a solid supporthaving a plurality of spatially distinguishable nucleic acids disposedthereon or therein. The nucleic acids can be disposed in an ordered orrandom pattern of features. An individual feature can be, for example, aspatially isolated nucleic acid molecule, or an ensemble of nucleic acidmolecules such as a cluster. An array can be a composite arraycomprising a plurality of individual arrays configured to allowprocessing of multiple samples. The individual arrays, referred toherein as “sub-arrays,” include groups of nucleic acid features.Sub-arrays appear in distinct regions with in a larger array. Thesub-arrays themselves can be ordered or non-ordered. Such sub-arrays canbe optionally spatially addressable. Sub-arrays can include clusters ofidentical nucleic acids. An example of a composite array composed ofindividual sub-arrays is a microtiter plate having wells in which theplate as a whole is an array of nucleic acids (or composite array) whileeach individual well represents a sub-array within the larger compositearray.

As used herein the term “nucleic acid member” means a single nucleicacid bound to a support that is part of an array and/or part of asub-array within a composite array.

As used herein the term “support” refers to a substrate for immobilizingan array of nucleic acids. A “support” is a material having a rigid orsemi-rigid surface to which a nucleic acid array can be attached or uponwhich nucleic acids can be synthesized and/or modified. Supports caninclude any resin, microbead, glass, controlled pore glass (CPG),polymer support, membrane, paper, plastic, plastic tube or tablet,plastic bead, glass bead, slide, ceramic, silicon chip, multi-wellplate, nylon membrane, fiber optic, and PVDF membrane.

A support can include any flat wafer-like substrates and flat substrateshaving wells, such as a microtiter plate, including 96-well plates.Exemplary flat substrates include chips, slides, etched substrates,microtiter plates, and flow cell reactors, including multi-lane flowcell reactors having multiple microfluidic channels, such as the eightchannel flow cell used in the cBot sequencing workstation (Illumina,Inc., San Diego, Calif.).

A support can also include beads, including magnetic beads, hollowbeads, and solid beads. Beads can be used in conjunction with flatsupports, such flat supports optionally also containing wells. Beads, oralternatively microspheres, refer generally to a small body made of arigid or semi-rigid material. The body can have a shape characterized,for example, as a sphere, oval, microsphere, or other recognizedparticle shape whether having regular or irregular dimensions. The sizesof beads, in particular, include, without limitation, about 1 μm, about2 μm, about 3 μm, about 5 μm, about 10 μm, about 20 μm, about 30 μm,about 40 μm, about 60 μm, about 100 μm, about 150 μm or about 200 μm indiameter. Other particles can be used in ways similar to those describedherein for beads and microspheres.

The composition of a support can vary, depending for example, on theformat, chemistry and/or method of attachment and/or on the method ofnucleic acid synthesis. Support materials that can be used in accordancewith the present disclosure include, but are not limited to,polypropylene, polyethylene, polybutylene, polyurethanes, nylon, metals,and other suitable materials. Exemplary compositions include supports,and chemical functionalities imparted thereto, used in polypeptide,polynucleotide and/or organic moiety synthesis. Such compositionsinclude, for example, plastics, ceramics, glass, polystyrene, melamine,methylstyrene, acrylic polymers, paramagnetic materials, thoria sol,carbon graphite, titanium dioxide, latex or cross-linked dextrans suchas Sepharose™, cellulose, nylon, cross-linked micelles and Teflon™, aswell as any other materials which can be found described in, forexample, “Microsphere Detection Guide” from Bangs Laboratories, FishersInd., which is incorporated herein by reference. A support particle canbe made of cross-linked starch, dextrans, cellulose, proteins, organicpolymers including styrene polymers including polystyrene andmethylstyrene as well as other styrene co-polymers, plastics, glass,ceramics, acrylic polymers, magnetically responsive materials, colloids,thoriasol, carbon graphite, titanium dioxide, nylon, latex, or TEFLON®.“Microsphere Detection Guide” from Bangs Laboratories, Fishers, Inc.,hereby incorporated by reference in its entirety, is a helpful guide.Further exemplary supports within the scope of the present disclosureinclude, for example, those described in US Application Publication No.02/0102578 and U.S. Pat. No. 6,429,027, both of which are incorporatedherein by reference in their entirety.

As used herein the term “light-induced degradation” means thelight-induced damage to one or more nucleic acids in an array of nucleicacids by exposure to illumination. Such degradation includes thecomplete or partial removal of individual nucleic acids from the supportto which the array is attached. For example, light-induced degradationcan include cleavage of the phosphodiester backbone at any of thenucleotides within an individual nucleic acid. Such degradation can alsoinclude removal of or reaction of a nucleic acid base or fluorescent tagcausing a loss in hybridization or fluorescence function. Light-induceddegradation can also include photo-induced crosslinking of nucleotides.The result of light-induced degradation can manifest as a decrease influorescence detection sensitivity in one or more regions or sub-arraysof an array nucleic acids when cycling through repeated detection steps,as might be observed, for example, when performing sequencing bysynthesis, sequencing by ligation and microarray scanning. When used inconjunction with the term “inhibiting,” this refers to a complete orpartial block in the extent of damage, for example, as can be quantifiedby the observed strength of fluorescent emission. Light damage can bemeasured, for example, as a function of fluorescence signal intensityversus number of repeated irradiation (detection) steps performed on thearray of nucleic acids. This process is sometimes referred to as Tintensity decay. Another assessment of light damage can be measured as afunction of error rate versus number of repeated irradiation (detection)steps performed on the array of nucleic acids.

As used herein the term “detection error rate” refers to a measure ofthe frequency of error in the identification of one or morefluorescently tagged nucleic acids in an array and/or sub-array ofnucleic acids. For example, when measuring fluorescence in a schemeemploying multi-color fluorescent tags, an error can arise bymisidentification of a tagged sub-array when, for example, thesignal-to-noise is eroded due to light-induced degradation of aplurality of nucleic acid members in the sub-array. Thus, the detectionerror rate is increased with the continual loss of individual nucleicacid members of an array or sub-array over numerous repeated cycles ofirradiation.

As used herein the term “irradiating” refers to exposing an array ofnucleic acids to illumination. The exposure can be for the purpose offluorescence detection, for example. Irradiation can be performed with alaser or similar light source. Irradiation can be performed over aselect section of the UV-visible spectrum and can employ one or morewavelength band filters. Irradiating can be performed over a period oftime to collect sufficient fluorescent emission data. The term “intense”when used in reference to illumination refers to the amount of powerthat is employed during irradiation of a portion of an array of nucleicacids. Intense laser irradiation include an amount between about 5milliWatts to about 500 milliWatts and a powerdensity in an amount frombetween about 1 to about 200 W/mm².

As used herein the term “detection solution” means a solution containingcompounds of the present invention that reduce light-induced degradationupon exposure of an array of nucleic acids to illumination. Thedetection solution is the solution that is used during a detection stepemploying irradiation.

As used herein the term “buffer,” when used alone refers to any otherbuffer solution not used as a detection solution. Buffer solutionsinclude those used in polymerase reactions, hybridizations, washing, orany other operation performed prior to the use of the detection solutionemployed in the invention.

As used herein the term “polyphenolic compound” refers to an aromaticcompound having multiple hydroxyl groups (i.e. phenolic groups) on abenzene or other aromatic ring. The benzene, or other aromatic ring, canbe optionally substituted with other substituents and/or fused rings.Exemplary polyphenolic compounds include, without limitation, gallicacid and lower alkyl esters thereof, monomethyl ethers thereof, andcombinations of lower alkyl esters and monomethyl ethers thereof,pyrogallol, and hydroquinones, such as t-butyl hydroquinone (TBHQ),2,4,5-trihydroxybutyrophenone (THBP).

As used herein the term “lower alkyl ester” refers to a C1-C6 alkylchain ester of a carboxylic acid. In some embodiments, a “lower alkylester” refers to a C1-C4 alkyl chain ester of a carboxylic acid.Representative esters include methyl, ethyl, propyl, butyl, pentyl, andhexyl esters. Any of the forgoing esters can be optionally branched.Such branched esters include iso-propyl esters, sec-butyl esters,iso-butyl esters and tert-butyl esters, for example.

In some embodiments, the present invention provides a method ofinhibiting light-induced degradation of nucleic acids during a detectionstep that includes irradiating the nucleic acids in the presence of adetection solution having a polyphenolic compound. The detectionsolution inhibits the amount of light-induced degradation of the nucleicacids.

In some embodiments, the present invention provides a method ofdetecting a nucleic acid having a fluorescent tag that includes a)irradiating the nucleic acid with light having a suitable wavelength toinduce a fluorescence emission; b) detecting said fluorescence emission;and c) repeating steps a) and b). The irradiating step is carried out inthe presence of a detection solution that includes a polyphenoliccompound. The detection solution inhibits light-induced degradation ofthe nucleic acid. In some embodiments, the detection solution includesgallic acid, a lower alkyl ester thereof, or mixtures thereof. In stillother embodiments, the detection solution includes a mixture of 1)gallic acid, a lower alkyl ester thereof, or mixtures thereof and 2) oneor more compound(s) selected from urea, ascorbic acid or salt thereof,and isoascorbic acid or salt thereof.

Methods of the present invention include a detection solution for useduring an irradiation step. The detection solution includes apolyphenolic compound which can be any aromatic system having two ormore phenolic hydroxyl groups, any one of which can also be a loweralkyl ether. Polyphenolic compounds can belong to any number ofstructural classes such as lignans, tannins, gallocatechins, andflavanoids, such as flavonols, flavones, catechins, flavanones,anthocyanidins, and isoflavonoids. Polyphenolic compounds of the presentinvention can exist in a glycosylated form with one or more sugarresidues attached to the polyphenolic compound. Such glycosylated formscan confer useful solubility properties in water, for example. Exemplarypolyphenolic compounds include, without limitation, apigenin,astragalin, aurantinidin, azaleatin, butin, caffeic acid, catechin,cyanidin, epicatechin, epigallocatechin, gallocatechin, gallic acid andlower alkyl esters thereof, pyrogallol, delphinidin, ellagic acid,eriodictyol, homoeriodictyol, europinidin, fisetin, ferulic acid,galangin, genistein gossypetin, hesperitin, hesperidin, hydroquinones,such as p-hydroquinone, t-butyl hydroquinone (TBHQ),2,4,5-trihydroxybutyrophenone (THBP), hydroxytyrosol, isorhamnetin,isosakuranetin, kaempferol, kaempferide, luteolin, luteolinidin,malvidin, myricetin, morin, naringenin, naringin, natsudaidain,pachypodol, pelargonidin, peonidin, petunidin, phloroglucinol,pinocembrin, poncirin, pterocarpans, pyrocatechol, quercetin,resorcinol, rhamnazin, rhamnetin, rosinidin, rutin, sinapyl alcohol,sakuranetin, sakuranin, sterubin, and tannic acid. In some embodiments,the polyphenolic compound can include gallic acid, a lower alkyl esterthereof, such as methyl gallate, ethyl gallate, propyl gallate,monomethyl ethers thereof, and combinations of lower alkyl esters andmonomethyl ethers thereof, or mixtures thereof. When employing gallicacid, its derivatives, or mixtures thereof, this component of thedetection solution can be present in a concentration ranging frombetween about 0.1 mM to about 200 mM.

Methods of the present invention employing a detection solution canfurther include one or more compounds to further reduce the amount oflight-induced degradation. In some embodiments these secondary compoundscan be selected from urea, ascorbic acid or salt thereof, andisoascorbic acid or salt thereof. Other secondary compounds that can beuseful to reduce light-induced degradation include, without limitation,diethylhydroxylamine, diethyldithiocarbamate, 2-phenyl-5-benzimidazolesulfonic acid, and bis-(2,2,6,6-tetramethyl-4-piperidyl)sebacate. Suchsecondary agents can be present in a range of concentrations frombetween about 10 mM to about 5M, including 10 mM, 20 mM, 30 mM, 40 mM,50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 200 mM, 300 mM, 400 mM, 500mM, 1M, 2M, 3M, 4M, and 5M, and any concentration in between.

In some embodiments, light-induced degradation can include oxidativedamage, such as disclosed in Cooke et al. FASEB J. 17 (10): 1195-1214(2003), which is incorporated herein by reference in its entirety. Forexample, light-induced degradation can include cleavage of thephosphodiester backbone at any of the nucleotides of a nucleic acid.Other degradation pathways can include the formation of8-oxo-2′-deoxyguanosine, the removal of or reaction of a nucleic acidbase or fluorescent tag causing a loss in hybridization or fluorescencefunction, and the photo-induced cross-linking of nucleotides.Degradation by reactive oxygen radical species such as hydroxyl radicalcan damage nucleic acids by hydrogen atom abstraction processes, such asfrom the methyl group of thymine or from the C—H bonds of2′-deoxyribose. Oxygen radical species can also oxidize nucleotides byreaction with unsaturations in nucleic acid bases, for example.

The detection solution having a polyphenolic compound can inhibitlight-induced damage of nucleic acids by various mechanisms. Withoutbeing bound by theory, the polyphenolic compound can act as a more rapidfluorescence quencher of the excited fluorophore than oxygen, thusreducing or preventing the formation of singlet oxygen as indicated inEquation 1:

As indicated in equation 1, singlet state excited fluorophore S₁ canreact preferentially with a polyphenolic compound (PP) in lieu ofreaction with oxygen, to provide a return of the fluorophore to thesinglet ground state S₀ while forming an excited state of thepolyphenolic compound (PP*). One skilled in the art will recognize thatthe return to ground state S₀ can alternatively proceed viaintersystem-crossing to a triplet excited state of the fluorophore T₁(not shown). This non-radiative pathway to the triplet state of thefluorophore can be promoted by interaction with the polyphenoliccompound. Ultimately, the energy transferred to the polyphenoliccompound can be effectively dissipated.

Alternatively, any singlet oxygen generated by fluorescence quenchingwith oxygen can react with the polyphenolic compound to preserve theintegrity of the nucleic acids. Similarly, any reactive oxygen speciessuch as superoxide anion or hydroxyl radical formation formed viamultiple photon absorption, as described above, can also be quenched bythe polyphenolic compound present in the detection solution. Yet anotherpossible mechanism by which the polyphenolic compound can reduce orprevent light-induced degradation is by intercepting and dissipating anyhigh energy UV-visible radiation generated by multiple photon absorptionby the fluorophore. In such a mechanism, the formation of reactiveoxygen species and/or the indirect pyrimidine dimerization caused byhigh energy emission is reduced or prevented. Finally, one skilled inthe art will appreciate that these high energy photochemical processescan operate in any combination.

As described above, inhibiting these light-induced degradation processesrefers to a complete or partial blocking of the extent of damage, forexample, as can be quantified by the observed strength of fluorescentemission with repeated exposure to intense illumination. Light-inducedegradation can be measured, for example, as a function of fluorescencesignal intensity versus number of repeated irradiation (detection) stepsperformed on the array of nucleic acids, such as in the sequencing bysynthesis examples described herein below. This process is sometimesreferred to as intensity decay. Another assessment of light-inducednucleic acid damage can be measured as a function of error rate versusnumber of repeated irradiation (detection) steps performed in an arrayof nucleic acids.

Still another assessment of light-induced damage can be to assay fornucleic acid degradation products. For example, measurement of8-oxo-2′-deoxyguanosine is commonly used to assess oxidative damage tonucleic acids. Damage can also be assessed by quantifying the degree ofthymine and cytosine homo- and heterodimerization. When employingdetection methods on solid support, the inhibition of light-induceddamage can be readily assessed by comparing arrays irradiated in thepresence and absence of the detection solution and measuring afluorescence signal intensity loss with repeated cycles of irradiation.

In some embodiments, methods of the present invention can be applied tonucleic acid detection in solution, while in other embodiments, methodsof the invention can be applied to nucleic acids attached to a support,such as in an array format. When employing nucleic acids on a support,the nucleic acids can be present in ordered or non-ordered arrays. Whenusing the detection solution having a polyphenolic compound, the amountof nucleic acids cleaved off a support can be reduced. Thus, in someembodiments, the light-induced degradation includes removal of a nucleicacid member from an array of nucleic acids.

When performing a detection step, irradiating the nucleic acids includesthe use of an appropriate light source to excite a tag, such as afluorescence tag. One skilled in the art will recognize that there arenumerous tags available and that the conditions of the irradiation, suchas choice of wavelength for irradiation and detection, will be guided bythe choice of tags being employed in the nucleic acid detection process.The tags can be the same for each type of nucleotide, or each nucleotidetype can have a unique tag. The tag is used in the identification of aparticular incorporated of nucleotide within the nucleic acid. Thus, forexample modified adenine, guanine, cytosine and thymine can all haveattached a different fluorophore to allow them to be discriminated fromone another readily. When sequencing on arrays, a mixture of tagged anduntagged nucleotides can be used. Suitable tags include, but are notlimited to, fluorescent tags, mass labels, magnetic labels and the like.By way of example, tags include biotin, dinitrophenol and fluorescein.Exemplary tagged nucleotides for use in the present methods aredescribed in WO 04/018497, and U.S. Pat. Nos. 7,541,444 and 7,057,026,which are incorporated by reference herein in their entireties.

Detectable tags such as fluorophores can be linked to nucleotides viathe nucleic acid base using a suitable linker. Suitable fluorophoresinclude those described in WO 2007/135368, which is incorporated byreference herein in its entirety. The linker can be acid labile,photolabile or contain a disulfide linkage. Exemplary tags and linkagesinclude those disclosed in WO03/048387. Other linkages, in particularphosphine-cleavable azide-containing linkers, can be employed in theinvention as described in WO2004/018493.

Irradiation is carried out with an irradiation source which can include,for example, a laser or other source to generate sufficient excitationlight intensity to produce detectable emission. The irradiation sourcecan be used in conjunction with any combination of wavelength filters.The irradiation step can be pulsed in some embodiments, or continuous inother embodiments. The intensity of a light source can be in a rangebetween about 5 milli Watts to about 500 milli Watts. The light sourcecan be a high-energy short arc-discharge lamp for example. Exemplarydischarge lamps include mercury burners, ranging in wattage from 50 to200 Watts, and the xenon burners that range from 75 to 150 Watts, andLEDs. The exact choice of light source intensity can depend on theextinction coefficient of the fluorophore. Larger extinctioncoefficients indicate that the absorption of a photon, also referred toas quanta, at a given wavelength region is more likely. The quantumyield denotes the ratio of photons emitted to photons absorbed, and isusually a value between 0.1 and 1.0. The quantum yield is a measure ofemission efficiency. Quantum yield values below 1 result from the lossof energy through non-radiative pathways, such as heat or aphotochemical reaction, rather than fluorescence emission. Extinctioncoefficient, quantum yield, the intensity of the light source, andfluorescence lifetime are all important factors that contribute to theintensity and utility of fluorescence emission.

Methods of the invention can include an irradiation step conducted in arange from about 360 nm to about 800 nm, with a light source havingpower in a range between about 5 to about 500 m watts. As describedabove, between about 360 to about 800 nm, light-induced degradation canbe ameliorated by the presence of the detection solution. Thisfacilitates the use of multiple fluorophores for simultaneousfluorescent emission detection. Thus, method of the invention can becarried out while incorporating the four common bases with differentialtags. The irradiation step can be conducted for a time period of about0.1 seconds to about 10 minutes. 175-500 ms on GA, 2 mm/s scan speed.Such irradiation times can include pulsed or continuous irradiation.

When performing multiple steps incorporating differentially labelednucleotides, methods of the invention can include replacing a solutionwith the detection solution prior to the irradiation step. For example,in sequencing by synthesis the solution used in adding the taggednucleotide can be replaced with the detection solution prior toirradiation. In other embodiments, the detection solution can be usedthroughout each of the steps in sequencing by synthesis, including washsteps, in addition to the detection step.

Fluorescent light emitted from the fluorophore can be detected at theappropriate wavelength using a suitable detection system such as, forexample, a Charge-Coupled-Device (CCD) camera, which can optionally becoupled to a magnifying device, a fluorescent imager or a confocalmicroscope. An alternate suitable detection system employs acomplementary metal-oxide-semiconductor (CMOS) detector. If sequencingis carried out on an array, detection of an incorporated base can becarried out by using a confocal scanning microscope to scan the surfaceof the array with a laser, to image fluorescent labels attached to theincorporated nucleotides. Alternatively, a sensitive 2-D detector, suchas a charge-coupled detector (CCD), can be used to visualize the signalsgenerated. This technique is particularly useful with single moleculearrays. Other techniques such as scanning near-field optical microscopy(SNOM) are available and can be used when imaging dense arrays. For adescription of scanning near-field optical microscopy, see Moyer et al.,Laser Focus World 29:10, 1993. An additional technique that can be usedis surface-specific total internal reflection fluorescence microscopy(TIRFM); see, for example, Vale et al., Nature, (1996) 380: 451-453).

When employing detection solutions of the invention to sequencing bysynthesis, for example, the method can include adding an additionalfluorescently tagged nucleotide to the array and repeating the detectionsteps each cycle. Methods of the invention employing repeated nucleotideaddition and detection steps can include at least 25 cycles in someembodiments, at least 75 cycles in other embodiments, and at least 100cycles in yet other embodiments. Methods of the invention includerepeating adding and detection steps for a number of cycles in a rangefrom between about 100 cycles to about 1,000 cycles, in someembodiments, from between about 100 cycles to about 500 cycles, in otherembodiments, and from between about 100 cycles to about 300 cycles, inyet other embodiments.

The ability to accurately sequence 25 or more, 50 or more, 75 or more,or 100 or more consecutive nucleotides in a sequencing reaction is asignificant advantage in applications such as genome re-alignment.

In some embodiments, an array of nucleic acids includes a primertemplate. Nucleic acid “sequencing-by-synthesis” involves sequentialaddition of one or more nucleotides to a growing polynucleotide chain inthe 5′ to 3′ direction using a polymerase in order to form an extendedpolynucleotide chain complementary to a template nucleic acid to besequenced. Exemplary sequencing methods are described, for example, inBentley et al., Nature 456:53-59 (2008), WO 04/018497; U.S. Pat. No.7,057,026; WO 91/06678; WO 07/123744; U.S. Pat. No. 7,329,492; U.S. Pat.No. 7,211,414; U.S. Pat. No. 7,315,019; U.S. Pat. No. 7,405,281, and US2008/0108082, each of which is incorporated herein by reference. Theidentity of the base present in one or more of the added nucleotide (s)is determined in the detection step. The identity of the added base canbe determined after each nucleotide incorporation step. The sequence ofthe template can then be inferred using conventional Watson-Crickbase-pairing rules. For the avoidance of doubt “sequencing” can alsoencompass incorporation and identification of a single nucleotide.Determination of the identity of a single base can be useful, forexample, in the scoring of single nucleotide polymorphisms.

The nucleic acid template for a sequencing reaction can include adouble-stranded region having a free 3′ hydroxyl group which serves as aprimer or initiation point for the addition of further nucleotides inthe sequencing reaction. The region of the template to be sequenced willoverhang this free 3′ hydroxyl group on the complementary strand. Theprimer bearing the free 3′ hydroxyl group can be added as a separatecomponent (e.g. a conventional oligonucleotide sequencing primer) whichhybridizes to a region of the template to be sequenced. Alternatively,the primer and the template strand to be sequenced can each form part ofa partially self-complementary nucleic acid strand capable of forming anintramolecular duplex, such as for example a hairpin loop structure.Nucleotides are added successively to the free 3′ hydroxyl group,resulting in synthesis of a polynucleotide chain in the 5′ to 3′direction. After each nucleotide addition the nature of the base whichhas been added can be determined, thus providing sequence informationfor the nucleic acid template.

Incorporation of a nucleotide into a nucleic acid strand (orpolynucleotide) refers to joining of the nucleotide to the free 3 ‘hydroxyl group of the nucleic acid strand via formation of aphosphodiester linkage with the 5’ phosphate group of the nucleotide.The nucleic acid template to be sequenced can be DNA or RNA, or even ahybrid molecule that includes both deoxynucleotides and ribonucleotides.The nucleic acid can include naturally occurring and/or non-naturallyoccurring nucleotides and natural or non-natural backbone linkages.

Nucleic acid templates to be sequenced can be attached to a solidsupport via any suitable linkage method known in the art. Linkage can bevia covalent attachment, for example. If the templates are “arrayed” ona solid support then the array can take any convenient form. Thus, themethod of the invention is applicable to all types of “high density”arrays, including single-molecule arrays and clustered arrays.

In some embodiments, the detection solution reduces a detection errorrate, when sequencing, by greater than 20% relative to a control lackingthe polyphenolic compound. The detection error rate is further reducedin the presence of one or more compound(s) selected from urea, ascorbicacid or salt thereof, and isoascorbic acid or salt thereof in thedetection solution. In further embodiments, the detection solutionreduces a detection error rate by greater than 40% relative to a controllacking the polyphenolic compound, which error rate is also enhanced bythe presence of one or more compound(s) selected from urea, ascorbicacid or salt thereof, and isoascorbic acid or salt thereof. In stillfurther embodiments, the detection solution reduces a detection errorrate by greater than 50% relative to a control lacking the polyphenoliccompound.

Error rates can be determined, for example, as described in Bentley, etal., “Accurate whole human genome sequencing using reversible terminatorchemistry” Nature 456:53-59 (2008). The error rate is the sequencingerror per cycle as determined by alignment of the phiX sequence againsta phiX standard genome using the ELAND algorithm that is part of thestandard pipeline analysis as described in the Pipeline User Guide(Illumina, Inc., San Diego, Calif.) and Bentley et al., Nature 456:53-59(2008).

In some embodiments, the present invention provides a method ofinhibiting light-induced degradation of nucleic acids during a detectionstep that includes irradiating a portion of the nucleic acids in thepresence of a detection solution that includes gallic acid, a loweralkyl ester thereof, or mixtures thereof. The detection solution reducesthe amount of light-induced degradation of the nucleic acids. The use ofgallic acid and its derivatives are shown below in Examples I and II.Gallic acid in its free form is capable of ionizing to carboxylate andits effectiveness as an inhibitor of light-induced degradation cantherefore be dependent on the pH of the system. By comparison, lowergallate esters can demonstrate a relatively constant effectiveness overa range of pHs.

Without being bound by theory, one skilled in the art will recognizethat a carboxylic acid group is modestly more electron withdrawing thanan ester. Thus, if gallic acid and related compounds are serving in anantioxidant role, one might expect an ester to perform slightly betterthan a carboxylic acid in reducing light-induced degradation. However,this benefit of gallate esters can be ameliorated by the need for watersolubility of the ester. In this regard, the ionizable carboxylic acidis useful to confer water solubility. As such, in some embodiments,detection solutions of the present invention provide gallic acid, loweralkyl esters of gallic acid, or mixtures thereof.

One skilled in the art will also recognize that other ester derivativesof gallic acid can be useful in the invention where sufficient watersolubility is conferred to the gallate ester. In some embodiments,additional solubility can be realized by use of a small amount of aco-solvent such as dimethyl sulfoxide (DMSO). By employing suchco-solvents in small quantities, the use of any gallate ester can beeffective in reducing light-induced degradation of nucleic acids. Insome embodiments, a water solublizing ester group can be employed, suchas a PEG ester of gallic acid. In such embodiments, the gallate estercan benefit from the improved antioxidant activity of an ester over acarboxylic acid, without sacrificing the useful water solubility of thecarboxylic acid functional group.

In yet further embodiments, the present invention provides a method ofinhibiting light-induced degradation of nucleic acids during a detectionstep that includes the nucleic acids in the presence of a detectionsolution that includes 1) gallic acid, a lower alkyl ester thereof, ormixtures thereof, and 2) one or more compound(s) selected from urea,ascorbic acid or salt thereof, and isoascorbic acid or salt thereof.This detection solution reduces the amount of light-induced degradationof the nucleic acids. As demonstrated in Examples I and II, urea hasshown a synergism in reducing light-induced degradation of nucleicacids. Unsubstituted urea, (NH₂)₂CO, is not generally classified as anantioxidant, and thus the role of urea in providing a synergistic effectin reducing light-induced degradation is not readily attributable toantioxidant activity. For example, it has been indicated that ureaaffords no apparent protection against reactive oxygen species atphysiological concentrations (Glazer, FASEB J. 2:2487-2491 (1988)).Moreover, it has been indicated that urea affords no apparent protectionagainst reaction with singlet oxygen (Dahl et al. Photochem. Photobiol.47(3):357-362 (1988)). Urea absorbs in the UV-visible regionsubstantially only below 245 nm. In this region of the UV spectrumpyrimidine dimerization is a significant pathway with respect tolight-induced nucleic acid damage. However, the role of urea inproviding additional protection against light-induced damage under theconditions of fluorescence detection, as provided in the Examples, arenot yet fully understood.

In some embodiments, the present invention is directed to a kit for usein accordance with the aforementioned methods. The kit generallyincludes one or more nucleotides, an enzyme capable of catalyzingincorporation of the nucleotides into a nucleic acid strandcomplementary to a nucleic acid template to be sequenced, and apolyphenolic compound suitable for preparing a detection solution. Thepolyphenolic compound can include gallic acid, a lower alkyl esterthereof, or mixtures thereof. In some embodiments, the kit furtherincludes a secondary compound selected from urea, ascorbic acid or saltthereof, and isoascorbic acid or salt thereof. The kit can furtherinclude buffer salts and a set of instructions for carrying outpreparation of a solution for use in fluorescence experiments.

Embodiments described herein can be used in a variety of known methodsand/or compositions for detecting nucleic acids, wherein light-induceddegradation of nucleic acids occurs or is suspected to occur. Forexample, the methods and compositions described herein are particularlyuseful when a detection method requires repeated or prolonged exposureof nucleic acids to light. In particular, detection of fluorescentlytagged nucleic acids is often used by irradiating a sample with lightthat has a suitable wavelength to induce a fluorescent emission from anucleic acids that contains a fluorescent tag. During the irradiationsteps of these methods, as described herein, damage to the nucleic acidscan occur. The repeated or prolonged exposure can result in a decreasein fluorescence detection sensitivity, which can manifest, for example,as an increase in detection error rates and reduced signal-to-noiseratios. Non-limiting examples of methods wherein the methods and/orcompositions for detecting nucleic acids that have repeated or prolongedexposure to light include high throughput or rapid sequencing techniquessuch as, sequencing by synthesis and sequencing by ligation, nucleicacid microarray detection techniques such as, gene chips and DNAmicroarrays, and quantitative polymerase chain reaction (Q-PCR)techniques such as, real time polymerase chain reaction (PCR) andmultiplex PCR.

One useful method for high throughput or rapid sequencing is sequencingby synthesis (SBS). SBS techniques that require repeated or prolongedirradiation of nucleic acids with light include, but are not limited to,the Genome Analyzer systems (Illumina Inc., San Diego, Calif.) and theTrue Single Molecule Sequencing (tSMS)™ systems (Helicos BioSciencesCorporation, Cambridge, Mass.). Briefly, a number of sequencing bysynthesis reactions are used to elucidate the identity of a plurality ofbases at target positions within a target sequence. All of thesereactions rely on the use of a target nucleic acid sequence having atleast two domains; a first domain to which a sequencing primer willhybridize, and an adjacent second domain, for which sequence informationis desired. Upon formation of an assay complex, extension enzymes areused to add dNTPs to a sequencing primer that is hybridized to firstdomain, and each addition of dNTPs is read to determine the identity ofthe added dNTP. This may proceed for many cycles. SBS techniques suchas, the Genome Analyzer systems (Illumina Inc., San Diego, Calif.) andthe True Single Molecule Sequencing (tSMS)™ systems (Helicos BioSciencesCorporation, Cambridge, Mass.), utilize labeled nucleotides to determinethe sequence of a target nucleic acid molecule. A target nucleic acidmolecule can be hybridized with a primer and incubated in the presenceof a polymerase and a labeled nucleotide containing a blocking group.The primer is extended such that the nucleotide is incorporated. Thepresence of the blocking group permits only one round of incorporation,that is, the incorporation of a single nucleotide. The presence of thelabel permits identification of the incorporated nucleotide. A pluralityof homogenous single nucleotide bases can be added during each cycle,such as used in the True Single Molecule Sequencing (tSMS)™ systems(Helicos BioSciences Corporation, Cambridge, Mass.) or, alternatively,all four nucleotide bases can be added during each cycle simultaneously,such as used in the Genome Analyzer systems (Illumina Inc., San Diego,Calif.), particularly when each base is associated with adistinguishable label. After identifying the incorporated nucleotide byits corresponding label, both the label and the blocking group can beremoved, thereby allowing a subsequent round of incorporation andidentification. Determining the identity of the added nucleotide baseincludes repeated exposure of the newly added labeled bases a lightsource that can induce a detectable emission due the addition of aspecific nucleotide base, i.e. dATP, dCTP, dGTP or dTTP. The methods andcompositions disclosed herein are particularly useful for such SBStechniques.

Another useful method for high throughput or rapid sequencing techniqueis sequencing by ligation. Sequencing by ligation is a well known methodfor sequencing that requires repeated or prolonged irradiation ofdi-base probes with light. Exemplary systems that use sequencing bysynthesis include the SOLiD™ system by Applied Biosystems (LifeTechnologies, Carlsbad, Calif.). Briefly, methods for sequencing byligation include hybridizing sequencing primers to adapter sequencesimmobilized to templated beads. A set of four fluorescently labeleddi-base probes compete for ligation to the sequencing primer.Specificity of the di-base probe is achieved by interrogating every 1stand 2nd base in each ligation reaction. Following a series of ligationcycles, the extension product is removed and the template is reset witha sequencing primer complementary to the n−1 position for a second roundof ligation cycles. Multiple cycles of ligation, detection and cleavageare performed with the number of cycles determining the eventual readlength. Sequencing by ligation methods have been developed by AppliedBiosystems in its Agencourt platform (see Ronaghi et al., Science281:363 (1998); Dressman et al., Proc. Natl. Acad. Sci. USA100:8817-8822 (2003); Mitra et al., Proc. Natl. Acad. Sci. USA100:55926-5931 (2003)).

In addition, the methods and compositions described herein can beparticularly useful for sequencing from an array of nucleic acids, wheremultiple sequences can be read simultaneously from multiple positions onthe array since each nucleotide at each position can be identified basedon its identifiable label. Exemplary methods are described in US2009/0088327; US 2010/0028885; and US 2009/0325172, each of which isincorporated herein by reference.

Other methods know in the art where repeated or prolonged irradiation ofnucleic acids with light include nucleic acid microarray detectiontechniques, such as gene chips and DNA microarrays. It is well know inthe art that reliability and consistency problems exist when scanningnucleic acid microarrays, particularly when scanning the same microarraymore than once. However, multiple scans can be required in order toobtain the full dynamic range of the labeled nucleic acids, for example,when using a microarray to determine gene expression levels. A singlescan attempts to capture the whole range of expression in the givensamples. This may not give the true picture of the expression of thewhole set of genes when a wide range of expression is present. Forexample, a gene in the sample may express as few as 200 copies, whereasa separate gene in the same sample may express 50,000 copies. In thisaspect, the methods and compositions described herein are particularlyuseful in maintaining the integrity of the nucleic acids over multiplescans.

Examples of nucleic acid microarray detection techniques known in theart include, but are not limited to, LabCard (ACLARA Bio Sciences Inc.,Santa Clara, Calif.); GeneChip (Affymetrix, Inc, Santa Clara, Calif.);LabChip (Caliper Technologies Corp, Hopkinton, Mass.); microarraysproduced by SurePrint technology (Agilent Technologies, Santa Clara,Calif.); a low-density array with electrochemical sensing (ClinicalMicro Sensors Inc., Pasadena, Calif.); LabCD System (Tecan Trading AG,Zurich, Switzerland.); Omni Grid (Gene Machines, Stillwater, Okla.); QArray (Genetix Ltd., Boston, Mass.); a high-throughput, automated massspectrometry systems with liquid-phase expression technology (GeneTraceSystems, Inc., Menlo Park, Calif.); a thermal jet spotting system(Hewlett Packard Company; Palo Alto, Calif.); Hyseq HyChip (Hyseq, Inc.,Sunnyvale, Calif.); BeadArray (Illumina, Inc., San Diego, Calif.); GEM(Incyte Microarray Systems, Fremont, Calif.); a high-throughputmicroarrying system that can dispense from 12 to 64 spots onto multipleglass slides (Intelligent Bio-Instruments, Waltham, Mass.); MolecularBiology Workstation and NanoChip (Nanogen, Inc., San Diego, Calif.); amicro fluidic glass chip (Orchid Cellmark, Inc., Dayton, Ohio); BioChipArrayer with four PiezoTip piezoelectric drop-on-demand tips (PackardInstruments, Inc., Meriden, Conn.)); FlexJet (Rosetta Inpharmatic, Inc.,Kirkland, Wash.); MALDI-TOF mass spectrometer (Sequenome, San Diego,Calif.); ChipMaker 2 and ChipMaker 3 (Arrayit Corporation, Sunnyvale,Calif.); and GenoSensor (Abbot Molecular, Des Plaines, Ill.) asidentified and described in Heller, Annu Rev. Biomed. Eng. 4:129-153(2002). Examples of gene chips or a microarrays are also described inU.S. Patent Publ. Nos.: 2007-0111322, 2007-0099198, 2007-0084997,2007-0059769 and 2007-0059765 and U.S. Pat. Nos. 7,138,506, 7,070,740,and 6,989,267.

Quantitative polymerase chain reaction (Q-PCR) techniques such as, realtime polymerase chain reaction (PCR) and multiplex PCR are well knownmethods of characterizing and quantifying nucleic acids. Such techniquesrequire repeated or prolonged exposure of nucleic acids to light,wherein the methods and compositions described herein are useful forinhibiting light-induce degradation. Five of the most popularchemistries for performing real-time PCR and/or multiplex PCR includeTaqMan® (Life Technologies, Carlsbad, Calif.), Molecular Beacons, FRETprobes, Scorpions® (Sigma-Aldrich, Inc, St. Louis, Mo.) and SYBR® Green(Life Technologies, Carlsbad, Calif.). All of these chemistries allowdetection of PCR products via the generation of a fluorescent signal.TaqMan® probes, Molecular Beacons, FRET probes and Scorpions® depend onFörster Resonance Energy Transfer (FRET) to generate the fluorescencesignal via the coupling of a fluorogenic dye molecule and a quenchermoiety to the same or different oligonucleotide substrates. SYBR® Greenis a fluorogenic dye that exhibits little fluorescence when in solution,but emits a strong fluorescent signal upon binding to double-strandedDNA.

TaqMan® probes depend on the 5′-nuclease activity of the DNA polymeraseused for PCR to hydrolyze an oligonucleotide that is hybridized to thetarget amplicon. TaqMan® probes are dual labeled oligonucleotides thathave a fluorescent reporter dye attached to the 5′ end and a quenchermoiety coupled to the 3′ end. Typically, TaqMan® probes consist of a18-22 bp oligonucleotide probe. These probes are designed to hybridizeto an internal region of a PCR product. In the unhybridized state, theproximity of the fluorescent reporter dye and the quench moleculesprevents the detection of fluorescent signal from the probe. Whenconducting a real time PCR experiment, a TaqMan® probe, complementary tothe target sequence is added to the PCR reaction mixture. During PCR,the probe anneals specifically between the forward and reverse primer toan internal region of the PCR product. When the polymerase replicates atemplate on which a TaqMan® probe is bound, the 5′-nuclease activity ofthe polymerase cleaves the probe. This decouples the fluorescent andquenching dyes and FRET no longer occurs. Thus, fluorescence increasesin each cycle, proportional to the amount of probe cleavage.

Like TaqMan® probes, Molecular Beacons also use FRET to detect andquantitate the synthesized PCR product via a fluorophore coupled to the5′ end and a quench attached to the 3′ end of an oligonucleotidesubstrate. A Molecular Beacon consists of 4 parts, namely a loop, stem,a 5′ fluorophore and a 3′ quencher. The loop is typically a 18-30 basepair region of the molecular beacon which is complementary to the targetsequence. The stem sequence lies on both ends of the loop and istypically 5-7 bp long. Both the stem sequences are complementary to eachother. The 5′ end of the Molecular Beacon contains a fluorophore and the3′ end of the molecular beacon contains a quencher dye that when thebeacon is in a closed loop shape, prevents the fluorophore from emittinglight. When a Molecular Beacon hybridizes to a target, the fluorescentdye and quencher are separated, FRET does not occur, and the fluorescentdye emits light upon irradiation. However, unlike TaqMan probes,Molecular Beacons are designed to remain intact during the amplificationreaction, and must rebind to the target in every cycle for signalmeasurement. Molecular beacons can report the presence of specificnucleic acids from a homogeneous solution. For quantitative PCR,molecular beacons bind to the amplified target following each cycle ofamplification and the resulting signal is proportional to the amount oftemplate.

FRET probes are a pair of fluorescent probes designed to hybridize toadjacent regions on the target DNA as described by Didenko,Biotechniques 31(5):1106-1121 (2001). Fluorophores are so chosen thatthe emission spectrum of one overlaps significantly with the excitationspectrum of the other. During PCR, the two different oligonucleotideshybridize to adjacent regions of the target DNA such that thefluorophores, which are coupled to the oligonucleotides, are in closeproximity in the hybrid structure. The donor fluorophore is excited byan external light source, then passes part of its excitation energy tothe adjacent acceptor fluorophore. The excited acceptor fluorophoreemits light at a different wavelength which can then be detected inspecific channels and measured. The light source cannot excite theacceptor dye.

With Scorpion® probes, sequence-specific priming and PCR productdetection is achieved using a single oligonucleotide as described inBates et al., Molecular Plant Pathology 2(5):275-280 (2001); Hart etal., J. Clin. Microbiol. 39(9):3204-12 (2001), and Thelwell et al.,Nucleic Acids Research 28(19):3752-61 (2000). Scorpion® primers arebi-functional molecules in which a primer is covalently linked to theprobe. The Scorpion® probe maintains a stem-loop configuration in theunhybridized state. A fluorophore is attached to the 5′ end and isquenched by a moiety coupled to the 3′ end. The 3′ portion of the stemalso contains sequence that is complementary to the extension product ofthe primer. This sequence is linked to the 5′ end of a specific primervia a non-amplifiable monomer. In the absence of the target, thequencher nearly absorbs the fluorescence emitted by the fluorophore. Inthe initial PCR cycles, the primer hybridizes to the target andextension occurs due to the action of polymerase. After extension of theScorpion® primer, the specific probe sequence is able to bind to itscomplement within the extended amplicon thus opening up the hairpin loopand separating the fluorophore and the quencher, which leads to anincrease in fluorescence emitted. The fluorescence can be detected andmeasured in the reaction tube during each successive cycle ofamplification.

SYBR® Green provides one of the simplest and most economical format fordetecting and quantitating PCR products in real-time reactions. SYBR®Green is an asymmetrical cyanine dye as described by Zipper et al.,Nucleic Acids Res. 32(12):e103 (2004). SYBR® Green preferentially bindsdouble-stranded DNA, but will stain single-stranded DNA with lowerperformance. The resulting DNA-dye-complex absorbs blue light (λmax=488nm) and emits green light (λmax=522 nm). Since the dye preferentiallybinds to double-stranded DNA, there is no need to design a probe for anyparticular target being analyzed. However, detection by SYBR Greenrequires extensive optimization. Since the dye cannot distinguishbetween specific and non-specific product accumulated during PCR, as anyPCR product accumulates, fluorescence increases. The advantages of SYBR®Green are that it is inexpensive, easy to use, and sensitive. Thedisadvantage is that SYBR® Green will bind to any double-stranded DNA inthe reaction, including primer-dimers and other non-specific reactionproducts, which results in an overestimation of the targetconcentration. For single PCR product reactions with well designedprimers, SYBR® Green can work extremely well, with spurious non-specificbackground only showing up in very late cycles. Similar cyanine dyes areknown in the art and include SYBR® Green II, SYBR Gold, YO (OxazoleYellow), TO (Thiazole Orange), and PG (PicoGreen).

TaqMan® probes, Molecular Beacons and Scorpions® allow multiple DNAspecies to be measured in the same sample, also known as multiplex PCR,since fluorescent dyes with different emission spectra may be attachedto the different probes. Multiplex PCR allows internal controls to beco-amplified and permits allele discrimination in single-tube,homogeneous assays. These hybridization probes afford a level ofdiscrimination impossible to obtain with SYBR Green, since they willonly hybridize to true targets in a PCR and not to primer-dimers orother spurious products. However, multiplex PCR will also requireadditional repeated and prolonged irradiation steps in order quantitatethe multiple fluorescent emissions from a single sample. Accordingly,the methods and compositions described herein are useful for inhibitinglight-induced nucleic acid degradation.

It is understood that modifications which do not substantially affectthe activity of the various embodiments of this invention are alsoprovided within the definition of the invention provided herein.Accordingly, the following examples are intended to illustrate but notlimit the present invention.

Example I Error Rate and 20 Cycle Intensity

This Example shows error rates with repeated detection cycles in asequencing by synthesis format using an eight lane flowcell. The data inthis example was generated using the standard procedure of setting up asequencing run on HiSeq instruments (Illumina Inc., San Diego, Calif.)according to the HiSeq UserGuide and using standard Illumina Sequencingreagents, except for the detection solution. Methods for the standardprotocols are available from Illumina, Inc. and referenced in Bentley etal., Nature 456:53-59 (2008), which is incorporated by reference hereinin its entirety. Briefly, a plurality of fluorescently labeled modifiednucleotides is used to sequence clusters of amplified DNA present on thesurface of a flow cell. To initiate the first sequencing cycle, one ormore differently labeled nucleotides, and appropriate reagents, e.g.,DNA polymerase, etc., are flowed into/through the flow cell by a fluidflow system. After incorporation, non-incorporated nucleotides arewashed away by flowing a wash solution through the flow cell. Adetection solution is then flowed through the flow cell while lasers areused to excite the nucleic acids and induce fluorescence. A deblockingreagent is then added to the flow cell to remove reversible terminatorgroups from the DNA strands that were extended and detected. Thedeblocking reagent is then washed away by flowing a wash solutionthrough the flow cell. The flow cell is then ready for a further cycleof sequencing starting with introduction of a labeled nucleotide as setforth above. The fluidic and detection steps are repeated several timesto complete a sequencing run.

To generate the detection solution used in the experiments, the testcompounds were added from powder to the detection solution to theindicated final solutions in Table 1 and the pH was checked and adjustedif needed. The “Image Cycle Pump” feature was used to test 3 compoundsper run such that lanes 1 and 2 were scanned in control detectionsolution, lanes 3 and 4 in detection solution containing a first testcompound, lanes 5 and 6 in detection solution containing a second testcompound and lanes 7 and 8 in detection solution containing a third testcompound. The intensity decay and error rates were compared between thelanes that received control detection solution and those that receiveddetection solution containing the test compounds.

Example II Error Over Cluster PF

This Example shows a gallic acid dose response using 20 (lanes 3 and 4)40 (lanes 5 and 6) and 80 mM (lanes 7 and 8) with lanes 1 and 2 beingthe no gallic acid controls.

This experiment was carried out exactly as described above in Example I.FIG. 3 shows a graph plotting error versus cluster passing filter (PF)number. The graph in FIG. 3 shows a significant dose-dependentimprovement in error rates (y axis) for all gallic acid concentrations.The graphs also shows an improvement in clusters passing filters with anoptimum of 40 mM—at 80 mM the number of clusters passing filter isreduced compared to 40 mM.

Example III Coverage Plots

This Example shows the errors per position in a reference sequence.

This experiment was carried out exactly as described in Example I. FIG.4 shows coverage plots for a control sequence, in the presence of gallicacid, in the presence of urea, and in the presence of gallic acid andurea, over 75 cycles. FIG. 5 shows coverage plots for a controlsequence, in the presence of gallic acid, in the presence of urea, andin the presence of gallic acid and urea, over 100 cycles. Coverage plotsanalyze error (Y axis) per position in a given genome (x axis, usingphiX genome as standard). The data shows that the phiX genome containshot-spots with significantly higher error rate than the rest of thegenome. Gallic acid in the detection solution reduces the error ratecertain hot spot significantly while not affecting other hot spot asmuch. Urea by itself has very little effecting reducing the error ratesbut show a remarkable synergy with gallic acid that almost eliminatesthe error in most hotspots and even lowers the error of the dominant hotspot (around position 4300) while each of the compounds alone have noeffect.

TABLE 1 Entry Concen- num- tration Error cycle20 ber Compound mM RateInt 1 1,4-Dihydroxy-2,6-dimethoxybenzene 10 0.81 73.91 21,4-Dihydroxy-2-Naphthoic Acid 10 5.22 64.32 32,2,6,6-Tetramethyl-4-piperidinol 10 3.11 50.47 4 2,3 Dihydroxy BenzoicAcid 50 0.63 78.86 5 3,5 Dihydroxy Benzoic Acid 10 0.62 75.66 6 3,4Dihydroxy Benzoic Acid 10 0.73 78.60 7 2-Phenyl-5-benzimidazole sulfonic30 0.97 79.28 acid 8 3,4,5-Trihydroxybenzamide 10 2.08 76.71 93,4-Diaminobenzoic acid 10 2.01 80.47 10 3-Aminobenzoic Acid 10 0.3375.59 11 3-Amino-4-Hydroxybenzoic Acid 10 1.66 80.72 12 3-HydroxybenzoicAcid 10 2.23 70.79 13 3-Hydroxy-4-Nitrobenzoic Acid 10 3.72 73.50 143-Hydroxyanthranilic Acid 10 0.85 81.59 15 3-HydroxyCinnamic Acid 101.49 76.84 16 4-Aminosalicylic Acid 10 1.75 80.08 17 4-Amino BenzoicAcid 10 0.57 74.85 18 4-Amino3-Hydroxybenzoic Acid 10 1.61 79.21 194-Amino-3-Methoxybenzoic acid 10 2.36 74.98 20 4-Hydroxybenzoic Acid 102.01 74.76 21 4-Hydroxy-3-Nitrobenzoic acid 10 3.09 75.91 224-Hydroxycinnamic Acid 10 1.92 68.78 23 5-benzoyl-4-hydroxy-2-methoxy 102.58 75.53 benzene sulfonic acid 24 1-Aza-3,7-dioxabucyclo{3,3,0]- 300.38 78.19 octane-5-methanol 25 Benzoic Acid 100 2.24 71.98 26 Bis[2,2,6,6-Tetramethyl-4- 20 0.92 76.17 piperidone] sebacate 27 CaffeicAcid 10 0.41 76.60 28 Catechol 10 1.58 78.54 29 Chlorogenic Acid 10 0.8679.65 30 1,4-Diazabicyclo[2,2,2]octane 10 1.02 76.32 31 Diaminotoluenesulfate 10 0.56 76.08 32 Diethyldithiocarbamate 25 1.39 76.28 33N,N-Diethylhydroxylamine 30 0.43 78.94 34 Diethyldithiocarbamate 10 1.4679.30 35 N,N-Diethylhydroxylamine 10 1.41 76.66 361,4-Dimethylpiperazine 10 1.03 76.17 37 Diethylenetriaminepentaacetic 101.28 68.53 acid 38 5-Ethoxysalicylic acid 10 0.50 75.31 39 Ethylgallate10 0.36 85.89 40 Ferulic Acid 10 0.77 72.58 41 Gallic Acid 100 1.3987.37 42 Gallic acid 200 0.82 78.83 43 Gallic acid 20 0.58 84.30 44Gallic Acid 50 0.39 84.98 45 Glutathione 10 0.75 74.70 46 Histidine 100.92 76.18 47 5-methoxysalicylic acid 10 0.76 73.57 48 Hesperidin methylchalcone 30 0.26 79.84 49 Hydrazine 10 0.63 75.32 50 Hydroquinone 100.92 79.48 51 Laurylgallate 10 1.69 65.94 52 α-Lipoic acid 10 1.01 76.3253 β-Mercaptoethylamine 10 1.75 72.29 hydrochloride 54 Melatonin 1 0.4372.42 55 Methyl 3,4,5-trihydroxy-benzoate 20 0.97 82.34 56 Methyl3,4,5-trihydroxy-benzoate 10 0.47 82.60 57 Methoxyhydroquinone 10 0.9077.17 58 N-AcetylCysteine 10 2.97 76.56 59 Nordihydroguaiaretic Acid 101.25 68.60 60 2Phenylbenzimidazole sulfonic 30 0.30 78.37 acid 61p-Coumaric Acid 10 1.63 77.05 62 1,4-Phenylenediamine 10 1.58 79.04dihydrochloride 63 Propylgallate 30 1.96 73.40 64 Phthalic acid 10 0.7373.97 65 α-(4-Pyridyl-N-oxide)N-tert- 10 1.72 70.99 butylnitrone 66Propylgallate 10 0.38 84.45 67 Pyridoxine 100 1.95 72.53 68 QuinicAcid100 1.79 68.36 69 Quercetin 1 0.50 77.43 70 Rutin 1 0.42 74.18 71Salicylate 10 0.64 76.49 72 Selenomethionine 10 3.34 62.84 73 SodiumSelenite 30 1.46 78.66 74 Sodium Sulfite 10 0.63 75.84 75 Spermine 1000.29 54.69 76 Sulfanilic Acid 10 1.57 76.47 77 Syringic Acid 10 1.1282.32 78 Tert-Butylhydroquinone 10 1.33 84.93 794-Hydroxy-2,2,6,6-tetramethyl- 10 1.97 71.31 piperidine 1-oxyl 80Terephthalic acid 10 0.86 74.54 81 Tetrachloro-1,4-benzoquinone 10 4.0354.01 82 2,4,5-Trihydroxybutrophenone 10 1.02 84.56 831,3,5-Tris(2-hydroxyethyl)isocyanurate 10 1.75 76.86 84 Thiourea 10015.31 51.62 85 2,2,6,6-Tetramethyl-4-piperidone 10 1.89 69.38hydrochloride 86 6-Hydroxy-2,5,7,8-tetramethyl- 10 0.84 74.97chroman-2-carboxylic acid 87 L-Tryptophan 10 1.69 76.44

TABLE 2 Entry Error cycle20 number Compound Rate Int 1 Ethylgallate 10mM + 10 mM Bis [2,2,6,6- 0.57 88.89 Tetramethyl-4-piperidone] sebacate 2Ethylgallate 10 mM + 10 mM 0.50 90.48 Diethyldithiocarbamate 3Ethylgallate 10 mM + 10 mM 0.49 69.01 N,N-Diethylhydroxylamine 4Ethylgallate 10 mM + 10 mM 0.35 70.58 2Phenylbenzimidazole sulfonic acid5 Ethylgallate 10 mM + 10 mM Pyrogallol 0.31 72.78 6 Ethylgallate 10mM + 25 mM NaAscorbate 0.34 85.64 7 Ethylgallate 10 mM + 30 mMHydroquinone 0.45 90.58 8 Ethylgallate 10 mM + 50 mM Gallic Acid 0.3487.25 9 gallic acid 100 mM + 250 mM mannitol 0.31 77.08 10 gallic acid100 mM + 2M Urea 0.17 80.90

Throughout this application various publications have been referenced.The disclosures of these publications in their entireties are herebyincorporated by reference in this application in order to more fullydescribe the state of the art to which this invention pertains. Althoughthe invention has been described with reference to the examples providedabove, it should be understood that various modifications can be madewithout departing from the spirit of the invention.

What is claimed is:
 1. A method of detecting a nucleic acid having afluorescent tag comprising: a) irradiating at least a portion of saidnucleic acid with light, wherein said light comprises a suitablewavelength to induce a fluorescence emission; b) detecting saidfluorescence emission; and c) repeating steps a) and b); wherein saidirradiating step is carried out in the presence of a detection solutioncomprising gallic acid, a lower alkyl ester thereof, or mixturesthereof, and further comprising urea, said detection solution inhibitinglight-induced degradation of said nucleic acid.
 2. The method of claim1, wherein said nucleic acid is in an array of nucleic acids attached toa support.
 3. The method of claim 1, further comprising adding afluorescently tagged nucleotide to said nucleic acid.
 4. The method ofclaim 1, further comprising replacing a solution with said detectionsolution prior to the irradiation step.
 5. The method of claim 2,wherein inhibiting light-induced degradation to said nucleic acidcomprises reducing the cleavage of a nucleic acid member from saidarray.
 6. The method of claim 1, wherein said gallic acid, said loweralkyl ester thereof, or said mixtures thereof is present in aconcentration ranging from between about 10 mM to about 200 mM.
 7. Themethod of claim 1, further comprising one or more compound(s), whereinsaid one or more compound(s) further enhances preservation of theintegrity of said nucleic acid.
 8. The method of claim 7, wherein saidone or more compound(s) is selected from ascorbic acid or salt thereof,and isoascorbic acid or salt thereof.
 9. The method of claim 1,comprising at least 50 cycles repeating step c.
 10. The method of claim1, comprising repeating said adding and detection steps for a number ofcycles in a range from between about 100 cycles to about 1,000 cycles.11. The method of claim 1, wherein the presence of said detectionsolution reduces a detection error rate by greater than 20% relative toa control.
 12. The method of claim 11, wherein one or more compound(s)selected from ascorbic acid or salt thereof, and isoascorbic acid orsalt thereof further reduce(s) said detection error rate.
 13. The methodof claim 1, wherein the presence of said detection solution reduces adetection error rate by greater than 40% relative to a control.
 14. Themethod of claim 13, wherein one or more compound(s) selected fromascorbic acid or salt thereof, and isoascorbic acid or salt thereoffurther reduce(s) said detection error rate.
 15. The method of claim 1,wherein the presence of said detection solution reduces a detectionerror rate by greater than 50% relative to a control.
 16. The method ofclaim 15, wherein one or more compound(s) selected from ascorbic acid orsalt thereof, and isoascorbic acid or salt thereof further reduce(s)said detection error rate.
 17. The method of claim 1, wherein saidirradiation step is conducted in a range from about 360 nm to about 700nm.
 18. The method of claim 1, wherein said irradiation step isconducted with a light source having power in a range between about 5 toabout 500 milliwatts.
 19. The method of claim 1, wherein saidirradiation step is conducted for a time period of about 0.1 seconds toabout 10 minutes.
 20. The method of claim 2, wherein adding anadditional fluorescently tagged nucleotide comprises using a polymeraseto add a single nucleotide.